PROPOSAL SUMMARY/ABSTRACT: Allergic diseases are characterized by the local tissue infiltration of unique inflammatory leukocytes that are often dominated by eosinophils. These infiltrating eosinophils are currently perceived as a single monolithic population, although recent studies suggest that unique eosinophil subtypes that potentially display distinct activities co-exist. Unfortunately, efforts to stratify eosinophils into functionally distinct groups have thus far proven to be difficult and time consuming, leaving the role of individual cells largely unknown. This proposal integrates growing definitions of eosinophil subtype-specific gene expression with a novel multi-RNA targeting approach, allowing single cell gene expression imaging within tissue biopsies by reiterative cycles of in situ hybridization. The central hypothesis of this proposal is that the immune polarization of tissue infiltrating eosinophils is disease specific, characteristic of ongoing local immune responses, and an indicator of disease activity. The immediate objective is to demonstrate the utility of this strategic in situ approach as a way to identify and image individual eosinophils within an available tissue biopsy or cell cytospin preparation. Strategically, these studies will initially use eosinophils and established mouse models of disease with which we have previously reported mechanistic studies exploring the role of eosinophils in these settings. Our intermediate goal is to translate these techniques into identifying eosinophil subtypes and their location in eosinophilic diseases with established immune characteristics. The long term goal is to translate this strategic approach to human subjects for identification of eosinophil subtypes in biopsies. The objectives will be accomplished by the completion of the following Specific Aims: (1) To define and standardize identification of eosinophil subtypes using our novel multi-biomolecule cleavable fluorescent probes for in situ single cell imaging of multiple mRNAs and/or proteins in cells and tissues. Our immediate objective will be to utilize our characterization of eosinophil-specific gene expression and the use of novel reiterative in situ hybridization strategies to image functionally distinct immune polarized eosinophil subtypes via a ?signature? pattern of gene expression; (2) To identify the immune polarized eosinophil subtypes resident in tissues linked with established mouse models of Th2 and Th1 human disease, including pulmonary and gastrointestinal diseases. The presence and location of eosinophil subtypes within tissues reflects a diversity of immune microenvironments in health and disease and, in turn, the importance of novel eosinophil-mediated activities. It is noteworthy that as part of these studies we will also define the distribution and location of eosinophil subtypes resident in healthy tissue. (3) To determine the utility of multiple-target in situ technologies to image human eosinophil subtypes using tissue sections/cell preparations from patients with Th2 and Th1 diseases characterized by eosinophilia.. This aim is expected to provide a proof-of-concept foundation that will be the base for future in situ stratification of leukocytes as markers of diagnostic and disease activity.